Apparatus for analyzing the optical properties of a sample

ABSTRACT

An apparatus for analyzing optical properties of a sample includes a housing to receive a light source and a detector; a sample locus defined relative to the housing and positioned such that when a light source and a detector are in predetermined positions, the sample locus is subject to illumination by the light source and the detector is positioned to receive and detect light from the sample; a cover on the housing, the cover being movable in a hinged manner between an open position and a closed position; and a sample-receiving surface for receiving a free-standing sample in liquid or semi-solid form. When the cover is moved to the closed position it encloses the sample locus, with the sample-receiving surface being tilted away from the horizontal during the closing movement and the sample being retained thereon by surface tension or adhesion and brought to the sample locus in an enclosed environment.

FIELD OF THE INVENTION

The present invention relates apparatuses for analysing the opticalproperties of a sample.

BACKGROUND OF THE INVENTION

The analysis of optical properties of samples using spectroscopicmethods is well known. Traditionally samples have been held in quartzglass cuvettes to standardise the sample volume through which radiationpasses, simplifying and standardising calculations.

More recently, the field of drop spectroscopy has come into prominence,allowing the spectroscopic measurement of microliter-scale volumes inliquid droplets. Due to the small volumes involved, which have highsurface areas relative to the liquid volume, it is important to isolatethe droplet quickly and reliably from the atmosphere. It is alsoimportant to optically isolate the sample, i.e. to enclose it in a darkvolume to avoid stray light and other radiation from interfering withmeasurements. At the same time, the very small volumes involved makehandling of the samples problematic, i.e. loading and unloading thedroplets from the measurement position.

WO 2012/140232 describes an instrument useful in such measurements,having a housing containing drop-supporting surface (or plinth) forreceiving a droplet of liquid. A cover is mounted on the housing, andthis receives a light source and provides communication between thelight source and the inner surface of the cover, allowing measurementsto be made on the droplet. The cover can be rotated to bring the lightsource into position over the plinth, or to reveal the plinth through anaperture for loading and removing samples, with the cover both rotatingand raising/lowering as it moves between positions to allow access tothe interior plinth on the one hand, and to seal the volume containingthe sample as well as bringing the measurement source into position onthe other hand. While this instrument addresses the problems associatedwith drop spectroscopy using this cover, it relies on a relativelycomplicated mechanism, and the necessity of installing the light source(or detector) in the cover imposes constraints on the kinds ofspectroscope that it can be used with. It would be advantageous toprovide an instrument that is compatible with third party spectroscopeswhile taking account of the particular challenges associated withhandling and measuring small droplets spectroscopically.

SUMMARY OF THE INVENTION

In accordance with one aspect of the invention there is provided anapparatus for analysing the optical properties of a sample, comprising:

-   -   a housing to receive a light source and a detector in        predetermined positions;    -   a sample locus defined relative to said housing and positioned        such that when a light source and a detector are in said        predetermined positions, the sample locus is subject to        illumination by said light source and the detector is positioned        to receive and detect light from the sample;    -   a cover on the housing, the cover being movable in a hinged        manner between an open position and a closed position, the cover        having an internal face which encloses the sample locus when the        cover is in the closed position;    -   a sample-receiving surface for receiving a free-standing sample        in liquid or semi-solid form, the sample-receiving surface being        disposed on the internal face of the cover such that when the        cover is in said open position the sample-receiving surface is        exposed to and accessible by a user in a generally horizontal        orientation for loading of a sample thereon;    -   wherein when the cover is moved to said closed position it        encloses said sample locus, with the sample-receiving surface        being tilted away from the horizontal during said closing        movement and the sample being retained thereon by surface        tension or adhesion and brought to said sample locus in an        enclosed environment.

As used herein, a “light source” is a source of any suitableelectromagnetic radiation, including visible, ultraviolet and infraredlight. The light source may be an optical port, window or fibre end thatconnects to a source externally of the housing or one that is locatedsomewhere else within the housing, and the detector may similarly be adetector port, window or fibre that feeds an external detector. Thepredetermined positions of the source and detector may be distinct orthe same position (such as when a fiber end is the interface to thesource and detector and the light is combined and split appropriatelyfrom the remote source and to the detector.

The detector may be any suitable detector to receive and detect lightfollowing an interaction with the sample, including light of the samewavelength(s) as the source (for example due to reflection, refractionor scattering by the sample) and light which is of a differentwavelength due to phenomena such as photoluminescence, fluorescence,Raman scattering, and surface enhanced Raman spectroscopy (SERS)including variants such as SLIPSERS. The light may be polarised toprovide information on the orientation of molecules in single crystalsand anisotropic materials, e.g. polarized Raman spectroscopy can providedetailed information as to the symmetry labels of vibrational modes.

The sample-receiving surface may be provided with nanoscale structuresto permit the enhancement of the Raman signal, such as by treating thesurface with suitable nanoparticles, and/or by providing a patternedsurface with nanoscale structures that permit the generation of surfaceplasmon resonance.

One design employs reusable sample dropheads or plinths, which can beprepared for SERS or other analyses. For example a stainless steelplinth is inert and reusable, and can be coated with colloidal gold, orsilver, or admixtures of gold and silver nanoparticles. After use, theycan be cleaned and reused for future measurements. Nanoparticles may bespherical or special shapes such as stars and irregular pointed shapesare useful because sharp points cause strong plasmonic emissions fromthese points. Other useful nanoparticles incorporate cores and shells.Such nanoparticles can be pipetted onto the plinth and the colloidalaggregation produces clustering of the particles and thus createhotspots.

Another option is to employ gold-plated or silver-plated plinths ordropheads, such as polystyrene dropheads plated with gold.

Furthermore, commercially available SERS substrates can be used, such asthe porous SERS membrane (SER-DM) available from iFyber of Ithaca, N.Y.,USA, or the gold-plated glass slides available from St. Japan ofDusseldorf, Germany, or the p-SERS inkjet printed substrates availablefrom Diagnostic anSERS, Inc. of Laramie, Wyo., USA or Metrohm AG ofHerisau, Switzerland, or the nanostructured, gold coated coppersubstrates available from Flew Solutions of Brisbane, Australia. (SER-DMand p-SERS are trade marks of the respective companies.)

Further options for SERS substrates usable as the sample-receivingsurface are to be found in the review article “Review of SERS Substratesfor Chemical Sensing”, Pamela A. Mosier-Boss, Nanomaterials (Basel).2017 June; 7(6): 142; doi: 10.3390/nano7060142. The sample locus may be(and in preferred embodiments actually is) smaller than the typicalsample size. In particular light from the source may be brought to afocus which is point-like or at least a small volume of high intensity.This means that the focal point may be positioned within and scannedrelative to the sample volume or the area of the sample-receivingsurface.

The apparatus of the invention provides a highly accessiblesample-receiving surface externally of the enclosure which aids in theaccurate loading of small droplets onto a small surface. Typicalsample-receiving surfaces have a diameter in the range of a fewmillimetres, such as 0.5 to 5 mm, with 1 to 2 mm plinths being suitablefor most applications. Such plinths can reliably hold volumes in themicroliter range, e.g. a 2 mm plinth can support by Laplace pressure a 3μl droplet. This reduces the likelihood of handling errors,contamination and sample loss. It further facilitates reliable and totalremoval of samples and cleaning of the sample-receiving surfacefollowing the measurement of one sample and in preparation for theloading of the next sample.

Samples in liquid form can be pipetted or otherwise deposited onto thesample receiving surface, and following measurement, the samplereceiving surface can be wiped clean with a tissue. Alternatively, thesample-receiving surface may be part of a disposable, replaceable orreusable plinth or drophead which can be swapped out and replaced with anew or cleaned member.

The hinged motion of the cover permits it to be flipped into the closedposition where it seals the enclosure and brings the sample intoposition for measurement. This mechanism greatly increases the designfreedom of the designer of the apparatus, allowing the cover to bemounted on almost any surface of a housing of any shape. This in turnmeans that the housing can be designed to receive a diverse range ofspectroscope assemblies, including a range of third party “boxes” suchas are conventionally sold and incorporated in laboratory instruments.Since the sample is held in position by surface tension, it isimmaterial that the final orientation of the sample-receiving surfacemay be e.g. vertical or (as in preferred embodiments) fully inverted.

The apparatus of the invention may be given additional versatility bypermitting the removal of a sample-receiving surface (such as a quartzplinth provided on a support) and its replacement by an alternativesample-receiving surface, or even in some cases by the substitution of aconventional sample holder into the position normally occupied by thesample-receiving surface.

By enabling the sample-receiving surface to be swapped out, one canprovide a range of plinths of different materials, sizes and properties.

The sample-receiving surface may also be swapped out to enable theinsertion of a standard material providing a known spectroscopicresponse, such as a polystyrene which exhibits sharp spectroscopic peaksat well-known wavelengths, or silicon having a Raman line at 520.7 cm-1.These materials can be used in bulk form, as a coating on a carrierplinth, or without swapping out the sample-receiving surface they couldbe powdered and used in a liquid droplet.

Preferably, in this regard, the sample holder is located in use within ahole in the cover, and when the sample holder is removed, the hole canbe accessed from the external cover surface to permit the insertion ofan alternative sample holder from the rear side thereof.

This feature will be described further below in relation to a vialholder that is positioned externally of the cover and which can hold avial that is exposed internally of the cover.

The sample holder may also be swapped out to be replaced with an opticalconnector to which an external probe could be attached. Such an externalprobe can allow the internal spectroscopic components to analyse asample that is outside or remote from the housing, e.g. by transmittingthe light e.g. laser light from the source, out through the opticalconnector that replaced the sample holder, and through an optical fiberwhich can be directed at the external sample. Light travels back to thedetector the same route. Such an external probe may be provided with acap or end member carrying an active coating such as is described inrelation to FIG. 18.

Preferably, the sample-receiving surface is translated through at least90 degrees as the cover moves from the open position to the closedposition.

More preferably, the sample-receiving surface is translated throughapproximately 180 degrees as the cover moves from the open position tothe closed position, with the sample-receiving surface being invertedwhen the cover is closed.

Preferably, the housing further comprises a support for receiving aspectroscopic assembly comprising a source and a detector mounted on aspectroscopic assembly housing, such that when said assembly is receivedby said support, the source and detector are in said predeterminedpositions.

In certain embodiments, the source may be coupled to an optical fiberterminating at a terminating surface through which the light is coupledfrom the source to the sample locus, the terminating surface beingpositioned such that when the cover is in a closed position, the sampleis brought into contact with the terminating surface.

Additionally or alternatively, the detector may be coupled to an opticalfiber terminating at a terminating surface through which the light iscoupled from the sample locus to the detector, the terminating surfacebeing positioned such that when the cover is in a closed position, thesample is brought into contact with the terminating surface.

Preferably, both the source and the detector are coupled to the sameoptical fiber having a single terminating surface which couples thelight into and out of the sample.

Preferably, said terminating surface carries active material thereonwhich enhances a spectral response.

Further, preferably, the terminating surface is provided with ananostructured treatment to realise a SERS response.

Preferably, the internal space comprises a sample chamber which isenclosed when the cover is in the closed position and which contains thesample locus, and an instrumentation chamber which receives the sourceand detector, the sample chamber being separated from theinstrumentation chamber by an impermeable wall having a wall sectionwhich is transparent to a wavelength of radiation emitted by the source.

Further, preferably, the sample chamber is sealed when the cover isclosed, and is sufficiently small in enclosed volume as to inhibitevaporation of a liquid sample droplet on said sample-receiving surface.

Further, preferably, the sample chamber comprises a reservoir for liquidwhereby the enclosed volume becomes saturated with vapour from saidliquid when said cover is closed.

Further, preferably, the sample chamber is provided with one or moreadditional environmental controls, such as a temperature controlmechanism.

Preferably, a sensor detects when the cover reaches a predeterminedposition moving between the open and closed positions, the sensor beingoperatively connected to a control circuit which prevents the sourcefrom being activated while the cover is open.

The sensor is advantageously a microswitch which is triggered as thecover approaches or reaches the closed position. It may be positioned toensure that the source remains inactive when the cover is opened beyonde.g. 5 degrees.

The apparatus preferably further comprises one or more motors operableto displace the sample-receiving surface, when the cover is in theclosed position, relative to the source and detector, thereby permittingfine adjustment of the material carried on the sample-receiving surfacerelative to the sample locus.

In this way, an apparatus having a source whose light is focussed to apoint or very small volume, can allow for fine tuning of theillumination relative to the sample. This has particular application inSERS applications, where the interaction of the sample with surfaceplasmons results in “hot spots”, i.e. locations showing significantlyenhanced response to the illumination.

Preferably, said one or more motors are operatively connected to a motorcontroller which is operable to scan the sample-receiving surface in acontrolled manner relative to the sample locus.

Preferably, said one or more motors include first and second motorswhich are disposed to displace the sample-receiving surface in mutuallyorthogonal directions in the plane of the sample-receiving surface.

Further, preferably, said one or more motors include a third motordisposed to displace the sample-receiving surface in a direction normalto the plane of the sample-receiving surface.

The addition of a z-control permits the position of a focal point to bevaried relative to the plane of the sample-receiving surface. This canbe used in scanning the sample at points other than at thesample-receiving surface, e.g. the bulk volume, the exposed surface, orabove the surface where vapours or gases may be given off. It alsopermits the apparatus to adjust for different sample heights ordifferent refractive indices which would otherwise cause the sample tofocus the source light away from the sample-receiving surface. As willbe apparent from what follows, the z-control may be provided in thesource optics or to translate the sample-receiving surface. The motorcontroller will preferably have control of such a z-direction motor ineither case.

Suitably, the apparatus may comprise in addition to said one or moremotors operable to displace the sample receiving surface, at least oneadditional motor operable to displace the source or an optical elementthrough which the light travels from the source to the sample locus,wherein the additional motor is operable to translate a focus of lightfrom the source in a direction normal to the plane of thesample-receiving surface.

Thus, for example, a pair of motors can displace the sample-receivingsurface in the x- and y-directions, and an additional motor can displacethe source to move the focal point of the incident light in thez-direction.

Thus, the motors can include x, y and z control (where x, y and x denotemutually orthogonal axes). This has particular advantages in permittingtargeted analysis of any point on the sample-receiving surface itself,as well as of any point within the bulk of the sample, the surface ofthe sample, or above that surface (which may be useful in the detectionof e.g. evaporating gases from a sample).

The motor controller can operate under manual control or it may beautomated. The automation of the motor controller can be pre-programmedto follow a pre-determined scan pattern, or it can be intelligent,whereby an algorithm seeks out a signal maximum or signal minimum forexample.

Preferably, the motor controller is under the control of a softwareprogram which conducts a pre-programmed coarse scan to identifypotential target areas, and which then performs a fine scan near one ormore signal maxima identified in the coarse scan, to locate a localmaximum.

Alternatively, the motor controller may be under the control of asoftware program which conducts a three-dimensional scan through thevolume of the sample, permitting the integration or aggregation of thedetected signal from a plurality of locations within the volume of thesample.

The apparatus may additionally comprise an optical sensing system toidentify the boundaries of the sample.

Preferably, the optical sensing system is provided as a camerapositioned to image the sample.

A preferred camera is micro-camera with visible and IR sensitivities. IRcapabilities are advantageous as such cameras are receptive towavelengths typically used in Raman/SERS applications.

The motor controller may be programmed to perform a scan within theboundaries identified by the optical sensing system. Those boundariescan be two-dimensional or three-dimensional.

Thus, the camera may image the sample-receiving surface, and the motorcontroller (or associated software) may derive from the image a set ofco-ordinates that define the limits of the sample-receiving surface. Thescan can then be conducted within the boundaries identified by thoseco-ordinates across the sample-receiving surface.

If a z-control is provided permitting either the sample-receivingsurface or the source (or source optics) to be translated in thez-direction normal to the sample-receiving surface, then the camera mayimage the sample volume and the motor controller (or associatedsoftware) may derive from the image a three-dimensional set ofco-ordinates that define the limits of the sample volume. The scan canthen be conducted within the boundaries of that sample volume.

Any suitable co-ordinate system can be used to define the scan area orscan volume, e.g. cartesian, polar, azimuthal, or a co-ordinate systemdefined in terms of motor steps for the individual motors.

The invention also provides a sample holder removal tool, the toolhaving a pair of jaws for gripping a sample holder body and providingclearance for a sample disposed on the sample holder body at a samplereceiving surface.

The invention further provides an apparatus for analysing the opticalproperties of a sample, comprising:

-   -   a housing to receive a light source and a detector in        predetermined positions;    -   a sample holder comprising a body having a sample-receiving        surface for receiving a free-standing sample in liquid or        semi-solid form, the sample-receiving surface being positionable        relative to the source and detector to permit analysis of a        sample thereon by said source and detector;    -   a receiving member, connected to the housing, for releasable        receiving the sample holder;    -   wherein the sample holder is removable from the receiving member        allowing its replacement by another sample holder.

Preferably, when the sample holder is received in said receiving member,a protrusion on the sample holder is accessible to grip the sampleholder and permit its removal.

Further preferably, the sample holder body has an exposed rim which isproud of the receiving member when the sample holder is in place andwhich provides said protrusion.

In a further aspect there is provided an apparatus for analysing theoptical properties of a sample, comprising:

-   -   a housing to receive a light source and a detector;    -   an environmental chamber body member in communication with the        source and the detector, such that a sample located in a        predetermined position relative to the environmental chamber        body member is positioned to be illuminated with light from the        source;    -   a carrier shaped to engage with the environmental chamber body        member to provide an enclosed environmental chamber when the        carrier and the environmental chamber body member are in        predetermined positions;    -   a sample-receiving surface for receiving a free-standing sample        in liquid or semi-solid form, the sample-receiving surface being        disposed on said carrier;    -   wherein the carrier is rotatably mounted on the housing such        that it can be rotated about an axis between a loading position        and a measurement position, with the sample-receiving surface        being exposed to a user when the carrier is in a loading        position, and with the carrier being positioned for engagement        with the environmental chamber body member when in the        measurement position.

In certain embodiments, the carrier is a cover having a hingedconnection to the housing, whereby the cover may be flipped about thehinged connection to move between the loading and measurement positions.

In other embodiments, the carrier is rotatable about an axis passingthrough the carrier to spin from the loading position to the measurementposition about said axis.

Preferably, the environmental chamber body member is movable within thehousing to engage with the carrier and provide an enclosed environmentalchamber containing the sample-receiving surface when the carrier is inthe measurement position.

Preferably, the environmental chamber body member is movable bytranslation from a first position to a second position, and when in thefirst position it is more distant from the carrier permitting thecarrier to spin around said axis, and when in the second position it isengaged with the carrier preventing the carrier from moving.

Embodiments can be provided where the sample-receiving surface istranslated into a measurement position without being rotated.

In another aspect there is provided a method of analysing a sample inthe form of a droplet provided on a sample-receiving surface, the methodcomprising:

-   -   providing a light source and a detector in a housing;    -   positioning said sample-receiving surface in or on the housing;    -   focussing an incident beam of light to a focal point in the        vicinity of the sample;    -   detecting light from the sample resulting from an interaction        with the sample, the sample-receiving surface, or the atmosphere        surrounding the sample;    -   measuring one or more parameters of the detected light;    -   translating the sample-receiving surface relative to the housing        such that the focal point is at a different region of the        sample, the sample-receiving surface, or the atmosphere        surrounding the sample; and    -   repeating the step of measuring one or more parameters of the        detected light following said translating step.

Preferably, said steps of translating the sample-receiving surface andrepeating the step of measuring the one or more parameters of thedetected light are performed multiple times, to provide a plurality ofmeasurements taken from different positions relative to the sample.

Further, preferably, the step of translating the sample comprisestranslating the sample in a plane parallel to the sample-receivingsurface.

Further, preferably, the step of translating the sample comprisestranslating the sample in two dimensions such that a plurality ofmeasurements are taken across said plane.

Further, preferably, the method further comprises the step oftranslating the sample-receiving surface in a direction normal to thesample-receiving surface and repeating a plurality of measurements inanother plane located at a different distance from the sample-receivingsurface.

Alternatively or additionally, the method may further comprise the stepof translating the focal point in a direction normal to thesample-receiving surface and repeating a plurality of measurements inanother plane located at a different distance from the sample-receivingsurface without translating the sample-receiving surface in a directionnormal thereto.

The method may comprise the step of analysing the plurality ofmeasurements to determine an optimal measurement position.

Preferably, the method comprises the step of performing a fine scan byrepeating the translation of the sample-receiving surface in thevicinity of said optimal measurement position to obtain an improvedmeasurement position.

Preferably, the sample-receiving surface is provided with nanostructuresenabling a SERS response, and the optimal measurement position providesa maximal SERS signal.

The method may comprise the step of analysing the plurality ofmeasurements by aggregating or integrating said plurality ofmeasurements.

The method may further comprise the steps of:

-   -   obtaining an image of the sample using an imaging device;    -   determining one or more sample boundaries from said image;    -   determining one or more translation limits within which the        sample-receiving surface is to be translated to thereby enable        said plurality of measurements to be taken from desired portions        of the sample, the sample-receiving surface or the atmosphere        surrounding the sample.

Optionally, the step of determining one or more translation limitsfurther comprises determining limits within which the focal point is tobe translated in a plane normal to the sample-receiving surface.

Preferably, the sample boundaries define a three-dimensional volume.

Preferably, the plurality of measurements are taken throughout thesample volume.

In a particularly preferred implementation, the plurality ofmeasurements comprise a series of measurements taken in a plane parallelto the sample-receiving surface, repeated at additional parallel planesin a plurality of slices through the sample volume.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further illustrated by the followingdescription of embodiments thereof, given by way of example only withreference to the accompanying drawings, in which:

FIG. 1 is a perspective view from a first angle of an apparatusaccording to the invention;

FIG. 2 is a perspective view from a reverse angle of the apparatus ofFIG. 1;

FIG. 3 is a perspective view of a sample holder assembly of an apparatusof the invention;

FIG. 4 is a perspective view of the assembly of FIG. 3 with the coveropen;

FIG. 5 is an exploded view of the FIG. 3 assembly;

FIG. 6 is a top view of the FIG. 3 assembly;

FIG. 7 is a sectional elevation of the FIG. 3 assembly taken along theline B-B in FIG. 6;

FIG. 8 is an enlarged detail of the sample holder with the cover of theFIG. 3 assembly closed;

FIG. 10 is a detail of the assembly of FIG. 3 from which FIG. 8 istaken;

FIGS. 10A-10C are respectively a side elevation, a perspective view fromabove, and a top plan view of a sample holder;

FIG. 11 is a perspective view of the sample holder assembly of FIG. 3with a SERS plate in place of the sample holder;

FIG. 12 is a sectional elevation through the assembly of FIG. 11;

FIG. 13 is an exploded view of the assembly of FIG. 11;

FIG. 14 is an exploded view of an alternative configuration of sampleholder assembly with a vial holder in place of the sample holder;

FIG. 15 is a perspective view of an alternative apparatus prior tomounting the sample holder assembly and the spectroscopic assembly;

FIG. 16 is an exploded view of a first spectroscopic assembly;

FIG. 17 is an exploded view of a second spectroscopic assembly;

FIG. 18 is a detail of a further embodiment;

FIG. 19 is a schematic illustration of a scanning technique forobtaining multiple measurements from a sample;

FIG. 20 is a schematic illustration of a further scanning technique forobtaining multiple measurements from a sample;

FIG. 21 is an embodiment adapted to perform scanning techniques such asare shown in FIGS. 19 and 20;

FIG. 22 is a view from above of a mechanism for translating a coverassembly;

FIG. 23 is a view from below of the FIG. 22 mechanism;

FIG. 24 is a sectional schematic view of a further apparatus with adifferent mechanism for bringing a sample into position for measurement;

FIG. 25 is a perspective view of a sample holder (plinth);

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In FIGS. 1 and 2, there is indicated, generally at 10, an apparatus foranalysing the properties of a liquid sample. The apparatus 10 comprisesan enclosure 12 defining an internal space 14 which receives aspectroscopic assembly 16 having a source and a detector behind anoptical port 18.

The spectroscopic assembly 16 is mounted on a cover plate 20 havingsecuring fasteners 22 which secure the cover plate to the housing. Thevertical (or Z) position of the cover plate and hence the spectroscopicassembly can be adjusted by a micrometer adjustment mechanism actingagainst a stop 26 on the housing, with slots 28 in the cover plateaccommodating a range of vertical positions.

An evaporation protection tray 30 can be mounted in the housing. Whencompartments in this tray are filled with a solvent (such as alcohol orwater) this acts as a reservoir to ensure that the interior of thehousing becomes saturated with vapour, which in turn inhibitsevaporation of a small volume sample exposed to the interior of thehousing. Adjustable feet 32 permit the apparatus to be accuratelylevelled.

A sample holder assembly 34 is mounted on top of the housing andcomprises a base plate 36 and a hinged cover 38, the base plate beingmounted in a recess 40 on the top surface of the housing. Theconstruction of various sample holder assemblies will be described inmore detail below.

FIG. 3 shows one embodiment of the sample holder assembly, which has thebase plate 36 and cover 38, the cover being mounted on a hinge 42 whichallows the cover to be lifted by a handle 44 and flipped backwards toreveal the underside or interior face of the cover and the area of thebase plate concealed by the cover. A pair of securing screws 46 areshown in FIG. 3 for securing the base plate to the housing. Also shownare a first micrometer adjuster 48 and second micrometer adjuster 50which permit fine adjustment of the cover position in X and Y directionsrelative to the base.

FIG. 4 shows the sample holder assembly of FIG. 3 when the cover 38 hasbeen flipped back through about 180 degrees about the hinge 42.Referring additionally to FIG. 5, this shows the same components, but inexploded view.

It can be seen from FIGS. 4 and 5 that the base plate 36 accommodates avapour tray 52. The vapour tray may render redundant a large vapour trayin the housing and it serves to provide a small enclosed volume withinwhich a sample will sit when the cover is closed. The vapour tray willbe described further below, but it suffices to note that it contains aglass window that exposes the sample to the optical components of thespectroscope.

The sample itself is not shown but FIG. 4 shows the sample holder 54 inexploded view. The sample holder is in the form of a stainless steelbody having a raised central disk, platform or plinth 56 which is sizedto receive a small volume of sample in the form of a liquid orsemi-solid (which term includes gels, pastes and slurries) volume thatis sufficiently small to be retained on the plinth by surface tension orLaplace pressure when the plinth is inverted (as when the cover isclosed).

The stainless steel sample holder 54 is received in a plinth holder 58that is mounted in the cover by a grub screw 60. A bore 62 (which is notfully visible in the view of FIG. 4 extends radially through the plinthholder 58 from the external annular surface to the internal annularsurface.

A retaining spring member 64 is located in the bore 60 prior to mountingthe plinth holder in position in the cover and securing it with the grubscrew. The retaining spring member has a cylindrical body with aninternal spring-mounted cylinder whose tip 66 extends from the inner endthereof such that in use it protrudes into the central hole of theplinth holder. The inner cylinder can be forced back into thecylindrical body of the retaining spring member 64 against springpressure, and in this way it serves to accommodate and secure the sampleholder 54 in the plinth holder 58.

This permits different sample holders such as sample holder 54 to beswapped into and out of the plinth holder 58 using a custom-designedtool 68 that is adapted to engage the rim of the sample holder withoutcontacting the central plinth or a sample loaded thereon.

It can be seen that the part of the hinge 42 which rotates with and isintegral with the cover has a recessed groove or cam surface 70 whichregisters with a micro switch 72 when the cover is closed. Opening thecover is thereby detected by the micro switch and this is connected tothe spectroscope to disable it. When the cover is closed the microswitch detects this and enables the operation of the spectroscope. Thisis an important safety feature when lasers are used in the spectroscope,and it can also protect sensitive detectors.

Also shown in FIG. 5 are a back cover plate 74 for covering the rear ofthe plinth holder and a vial holder clamp 76. The back cover plate 74may be removed, along with the plinth holder 58 and the sample holder54, and an alternative form of sample holder, such as a vial, to bemounted to the vial holder clamp, in a modified form of operation.

FIG. 6 shows a top plan view of the sample holder assembly of FIGS. 3-5(with internal features shown in broken lines), and FIG. 7 shows thecross-section through FIG. 6 taken along the line B-B.

It can be seen that with the cover 38 in its closed position the plinthholder 58 is brought face-down onto the vapour tray 52.

FIGS. 8 and 9 show the detail of this arrangement in enlarged form, withFIG. 8 being a detail of FIG. 9. The vapour tray 52 has an internalannular moat 78 and a raised central cylindrical wall 80 that definesthe internal wall of the moat 78. The moat can be filled with a solvent(such as the solvent used in a droplet of sample (not shown) positionedand retained on the central plinth 56), and this promotes a saturatedatmosphere to inhibit evaporation of the droplet in the very smallconfined volume between the cover and the vapour tray when the cover isclosed.

The plinth is positioned centrally over the cylindrical wall 80, whichin turn is aligned vertically with the optical port of the spectroscope,allowing light to illuminate the sample and for light from the sample(e.g. reflected, refracted or emitted radiation) to be collected. Theunderside of the vapour tray is separated from the rest of the interiorof the housing by a transparent (e.g. quartz glass) disc 82 to maintainseparation of the atmospheres while maintaining optical continuity. Itwill be appreciated that such a cover need only be transparent towavelengths of interest, which may vary according to the type ofanalysis being conducted and the wavelengths used (e.g. infrared,visible or ultraviolet spectroscopy, Raman spectroscopy or fluoroscopicmeasurements). A sample located on the plinth, in this way, is broughtto a predefined sample locus where measurements can be taken reliablyand accurately using the source and detector which are also inpredetermined positions.

FIGS. 10A-10C show an alternative form of sample holder 54′. As with theholder shown in FIGS. 4-9, a central raised plinth 56 is provided toreceive a sample thereon. Also as in the earlier embodiment, a rim 84extends peripherally from the top surface 86 and this allows engagementof the sample holder by the tool 68 (FIG. 4). Unlike the sample holderseen in FIG. 4, however, the sample holder 54′ of FIGS. 10-12 has acircumferential V-shaped recess 88 around its cylindrical surface. Thisrecess can engage with the retaining spring member 64 of FIG. 4regardless of the rotation of the sample holder when it is inserted intothe plinth holder. In contrast the sample holder 54 of FIG. 4 can beseen to have a specific recess positioned on its cylindrical surface,requiring it to be inserted with the correct orientation to be properlyretained.

While the sample holders thus far described are machined from a singlepiece of stainless steel, alternative materials can be used. Inparticular, quartz and ceramic holders are very suitable. Such holderscan be made entirely from an alternative material, or for example aquartz or ceramic rod having the diameter of the plinth only may beembedded centrally in a larger sample holder body made from any suitablematerial (such as metal, glass or plastic).

The provision of the sample holder (whether of FIGS. 4-9 or of FIGS.10A-100 or any alternative version) on the internal surface of thecover, and the flipping motion of the cover whereby it can be moved in ahinged manner between closed and opened positions, provides significantadvantages. It enables small (microliter-sized) droplets and samples tobe accurately and easily positioned on a very small target area with thetarget area exposed and fully accessible

FIGS. 11-13 shows an embodiment similar to that of FIGS. 4-9 in theoperation and movement of the cover, but in which the sample holder is aSERS plate 90 carried by the cover instead of the sample holder 54 or54′. SERS (or surface enhanced Raman spectroscopy) is a technique thatenhances Raman scattering by molecules adsorbed on rough metal surfacesor customised nanostructures. The enhancement factor can be as much as10¹⁰ to 10¹¹, which opens up the possibility of single moleculedetection.

SERS arose out of the observation that a roughening of the surface themolecules are adsorbed on helps enhance the Raman process, which in facthas proven to be a result of enhancement of the local electromagneticfield close to the roughened surface due to the excitation of alocalized surface plasmon, and chemical mechanisms giving rise tofurther enhancement for molecules adsorbed onto specific sites(hot-spots) where the resonant charge transfer occurs. The resultingenhancements occur most commonly when the excitation laser frequencycoincides with a localized surface Plasmon resonance (LSPR) of aplasmonic-active material such as Au, Ag and Cu, over normal Ramanscattering, making it a very powerful technique for sensing, as it alsooffers label-free detection of biomolecules in their naturalenvironment. In the study of biological species, SERS is often coupledwith resonance Raman by using a laser excitation wavelength in resonancewith the molecular electronic levels (SERRS).

The complexity of SERS, particularly in relation to samplepreparation—that may also factor in the SERS background—is a currentbottleneck to realisation of its full potential. SERS has been shown tobe capable of single molecule detection with high specificity, butsingle-molecule sensitivity in diluted solution is a challenge whichlimits its application, particularly in forensics and healthcarediagnostics, where for example, early warning focuses on smallconcentrations of biomarkers in biofluids such as blood. SERS depends onthe statistical binding of analytes to the SERS hot-spots where theelectromagnetic field is particularly intense. The creation of suchhot-spots is a highly-active area of research in the field, with variousapproaches to nanofabrication being taken, including top-downlithography-based fabrication, and bottom-up wet chemistry of structureson surfaces. In the liquid phase, molecules can be dispersed far awayfrom the surface, making an encounter of the molecules with hot-spotsstatistically unlikely. Approaches to overcoming this problem includethe introduction of plasmonic nanoparticles into the solution,functionalisation of nanoparticles with ‘grabby’ bioreceptors in thecase of biomolecules, the use of microfluidic channels, nanoporousmembranes, super-hydrophobic surfaces and external—both electrical andoptical—stimuli to guide and concentrate the molecules towards thefabricated hot-spots. Some approaches use a combination of techniques toincrease the possibility of an analyte interaction with a hot-spot.

The plate 90 is prepared in advance with a suitable surface treatment,such as gold or silver nanoparticle colloidal preparations. The sampleis positioned on the SERS surface, and measurements are taken of thesurface enhanced Raman spectrum. In an alternative modification, theSERS plate can be mounted on the base plate when the cover is opened toreveal the base plate. A raster scanning mechanism can be provided tomove the illumination spot from a laser in the spectroscope (whether byphysically moving the laser, or the spectroscope assembly, or byoptically scanning the position from a fixed laser in known manner). Inthis way the return signal can be detected to determine a maximum whenthe illumination spot is targeted to a point of maximum SERS activity onthe SERS plate, and this can be used as a pre-calibration step.

FIG. 14 shows a modification to the embodiment of FIGS. 3-9, in whichthe sample holder 54, plinth holder 58 and back cover 74 have beenremoved and in their place a vial holder 91 has been mounted, using thevial holder clamp 76, to the outside of the cover 38, with asample-containing vial 92 exposed to the interior of the cover. Aprotective cap 94 is provided on the exterior of the vial holder 91.This modification adapts the instrument for measurement of samples invials rather than in the form of droplets or small samples on asample-receiving surface.

FIG. 15 shows an alternative embodiment of apparatus from FIG. 1, withthe sample holder assembly 34 of FIG. 14 ready to be mounted in place.It will be appreciated that this is the same overall sample holderassembly as in FIGS. 3-9 in terms of its construction, base plate andcover, and only the vial holder differentiates it from the FIGS. 3-9embodiment. The sample holder assembly 34 is mounted in place onto arecess 96 on the top surface 98 of a housing 100. A removable accessplate 102 enables a user to access the interior for removal andreplacement of a spectroscopic assembly 104 (shown outside the housing).As in FIGS. 1 and 2, this assembly 104 is mounted on a mounting plate106 which can be fixed into position within the housing with brackets108 (FIG. 16) and thereby correctly position the assembly 104 relativeto the sample locus. A spacer 110 adapts the position of a particularspectroscopic assembly to the mounting plate 106, and it will beappreciated that different instruments can thereby be substituted intothe correct position.

The spectroscopic assembly pictured is a CBEx handheld Ramanspectrometer (produced by Snowy Range Instruments of Laramie, Wyo.)which provides a spectral range of 400 to 2300 cm-1 from a 70 mW laserthrough an optical port 112, with the ability to perform rasterscanning. Any other suitable spectroscopic instrument can be used.

FIG. 17 shows a fibre optic Raman probe 114 which is connected by fibreoptic cable 116 to an external spectroscope (not shown), the opticalsignals being emitted and received by an optical port 118 which acts assource and detector. As with the embodiment of FIG. 16, the probe 114 ismounted on a plate 120 which is mountable within the housing 100 of theFIG. 15 apparatus. Both the FIG. 16 and FIG. 17 embodiments have amicrometer adjustment mechanism 122 for varying the vertical Z positionof the spectroscopic assembly relative to the housing.

The above embodiments describe a free-space optics implementation, wherethe source is remote from the sample, and light is transmitted to thesample from the source and from the sample to the detector within theapparatus.

FIG. 18 shows a detail of an alternative embodiment, in which the sourceand detector (not shown) are each coupled to an optical fiber 130, whichterminates at a terminal surface 132. The terminal surface 132 ispositioned relative to the sample-receiving surface 134 (shown when thecover 138 is closed) such that the sample 136 makes contact with theterminal surface 132. The contact may be with the surface of the sampleas shown or the terminal surface may be positioned such that it isimmersed in the sample.

The arrangement couples the light directly into and out of the sample.The terminal surface 132 may be treated with a surface treatment thatprovides an enhanced response, such as a nanostructured coating ortreatment that provides a SERS response. The terminal surface or someother part of the optical fiber that is immersed in the sample may becoated with a reagent or active agent that causes a chemical reaction ora biological response, enabling the sample to be analysed by generatingsuch a reaction or response and analysing a spectroscopic characteristicof the resultant species.

FIG. 18 shows the terminal surface as integral with the optical fiber.The terminal surface may alternatively be provided on an end member orcap that is placed on the optical fiber and is transparent to afrequency of interest. This permits the cap to be easily swapped in andout, which may be advantageous to enable different responses andreactions to be studied, or to avoid cross-contamination betweensamples.

FIG. 19 shows a schematic illustration of a scanning capability that maybe incorporated in the apparatuses described herein. A camera 150 ispositioned to image a sample 152 (in this case a droplet) positioned onthe sample-receiving surface of a plinth 154. It will be appreciatedthat the sample is shown sitting on top of the surface but in theembodiments described above this arrangement is typically inverted sothat the droplet hangs upside down from the sample-receiving surfacewhen the cover is in the closed position. Light 158 from a source 156 isfocussed (by the optical properties of the source and the refractiveproperties of the droplet) to a point 160 on the surface where thedroplet sits.

By translating the sample-receiving surface in two dimensions, e.g.using an x- and y-raster scanning mechanism, the detected signal may beintegrated across the surface, or a search may be performed for anoptimum signal position. In the case of SERS measurements, where theresponse may increase by several orders of magnitude at points where theexcitation frequency coincides with a localized surface Plasmonresonance (LSPR) of a plasmonic-active material such as Au, Ag and Cu,there may be localized hot spots that provide greatly increased signalstrength.

Accordingly, the surface may be translated through a scanning pattern ofany suitable design (for example an x-y raster pattern, an orbitalpattern or any other suitable pattern), to identify areas of increasedresponse, using feedback from the detector. This is preferably done as acoarse search, followed by a fine scan in one or more areas ofparticularly increased signal strength.

Thus, in an automated fashion, an initial planar raster scan of theplinth surface is undertaken at the drop base centre, out to the dropedge. The software monitors and stores the Raman spectrum at each x, yposition, and that with maximum intensity Raman signal is identified forthe scan area.

The obtained Raman spectra can be used individually, integrated oraveraged over the full drop layer.

The camera image is provided to a controller which sets the scanparameters, by extracting from the image the boundaries of the surface,to assist in ensuring that the scan covers the desired area. As analternative, the camera could be omitted and the boundaries could beprogrammed into the system or detected by the detector (e.g. a sharpdrop-off of signal at the edge of the droplet).

FIG. 20 shows a schematic illustration of a three-dimensional scanningarrangement in which the same parts are denoted by the same referencenumerals as in FIG. 19. In addition to motor controllers or manualcontrols that enable translation of the sample-receiving surface in thex-y plane as in FIG. 19, the sample-receiving surface may also betranslated in the orthogonal z-direction. (Alternatively, the source orsource optics could be translated to move the focal point 160 relativeto the sample-receiving surface of the plinth 154.

As indicated by the “slices” 162, the volume of the sample may bescanned completely by performing scans across the sample volume inplanar regions separated by a small z-distance. Thus, a planar scan canbe taken along the plinth surface, out to the edges of the drop. Theprocedure is repeated at fixed Z-offset distances, out to the upper edgeof the drop; a z-axis motor offsets the z-axis position of the plinth(or of the source or source optics to translate the focal point to a newz-position), and the plinth is scanned in the next x-y plane a fixeddistance offset in z, within the drop volume limits.

The skilled person will appreciate that with x-, y- and z-controls,alternative scanning modalities are also enabled, such as scanningvertical slices (e.g. x-z plane or y-z plane) and then translating theplinth laterally to shift the plane where the slices are scanned, or inany other suitable scan pattern. Using the embodiment of FIG. 20, theapparatus may provide measurements from any point on thesample-receiving surface, or any point within the droplet volume bulk,or on the exposed droplet surface, as well as in the atmospheresurrounding the droplet. This can be used to determine areas of signalmaximum, or to integrate the signal across the droplet, or to analysethe surface or surrounding atmosphere, depending on the analytical goal.

Thus, the stored Raman spectra that have been taken at each point (orpredetermined points assigned in software, for example, spectrum withmaximum intensity Raman signal per scan layer) in each planar scan canbe integrated or averaged into one Raman spectra for the drop volume.

The camera is again optional but a preferred addition to the apparatus,in order to determine the extent of the droplet volume. Imagerecognition software can extract the position of the droplet surface andthis can be translated to co-ordinates defining the boundaries of thescan (noting that the scan may extend outside the droplet in cases whereanalysis of gaseous components from the droplet are of interest). Theuse of a camera also enables the droplet volume to be calculated whichmay assist in other calculations, such as in determining refractiveindex, surface tension, and so on.

FIG. 21 illustrates one embodiment for realising the scanningfunctionality described in relation to FIGS. 19 and 20. The embodimentof FIG. 21 is identical to that of FIG. 3 with the exception of thespecifically identified changes now discussed. In place of themicrometer adjuster mechanisms 48 and 50 of the FIG. 3 embodiment, x-yadjustments are enabled by a first motorised actuator 170 allows for anx-direction translation of the flipping mechanism as a whole, a secondmotorised actuator 172 enables translation in the y-direction, and athird motorised actuator 174 enables translation in the z-direction.Each actuator is driven by a respective motor 176, with the motors underthe common control of a motor controller (not shown) which may translatethe entire cover mechanism and therefore the sample-supporting surfaceto any position within the limits of travel defined by the threeactuators. Motor controller software may thus realise the scanningfunctionality described above, outputting appropriate control signals tothe individual motors.

The third motorised actuator may be omitted if z-scanning functionalityis not required in simpler apparatuses. Alternatively, it may beprovided on the optical components to scan the focal point of theincident light relative to the sample-supporting surface. However, it ispreferred to include a z-motor on the optical components or thecover/sample-supporting surface in order to increase the utility of theapparatus, permit better focussing (or defocussing where desired) andenable volumetric scanning.

The motors 176 in this embodiment (which is an example, and which theskilled person may vary according to design needs by using differentmotors and mechanisms) are stepper motors with 2045 steps perrevolution, driving the actuators via M3×0.5 (ISO standard) threadedrods. Thus, a resolution of 244 nm per step is achieved. If such aresolution is not needed, then for example a stepper motor with 200steps per revolution could be used, giving a resolution of 2.5 μm perstep. The travel of each actuator is a minimum of 10 mm, so that a scanvolume of 10 mm×10 mm×10 mm is achieved, enabling any part of a samplelocated in such a volume to be analysed.

Each stepper motor is driven by a respective stepper motor PCB. A pairof micro switches (not shown) detect end position of travel of the coverassembly. A Raspberry Pi single board computer provides control signaloutputs to the PCBs, according to programmed control instructions whichmay receive as inputs the image from the camera (if present) and fromthe detector. Thus, the program control may hunt for a signal maximum ina predefined scan pattern or it may perform a full-surface 2D scan or afull-volume 3D scan according to the needs of the user.

The skilled person will readily appreciate that alternative controlsystems may be provided.

As an alternative to stepper motors, linear motors could be employed, orpiezometric inertial-slider (slip-stick) motor stages that can beactuated individually or together by application of a periodicexponential voltage to the piezoelectric elements on respective x-, y-and z-stages.

An exemplary mechanism for enabling the cover assembly to be translatedis illustrated in FIGS. 22 and 23. FIG. 22 shows the mechanism fromabove and FIG. 23 from below. In FIG. 22 there can be seen an outerframe 180 which is mounted to the housing of an apparatus (not shown). Afirst slider 182 is slidably mounted within the frame, and a secondslider 184 is slidably mounted within the first slider 182. The flippingcover mechanism (not shown) is secured to the second slider 184, suchthat the sample-receiving surface locates within an aperture 186 in thesecond slider when the cover is in the closed position.

Referring to FIG. 23, a first stepper motor 188 is mounted on the frame180 and is connected to the first slider 182 so as to permit thecontrolled translation of the first slider relative to the frame. Asecond stepper motor 190 is mounted on the first slider 182 and isconnected to the second slider 184 to permit the controlled translationof the second slider relative to the first slider. By appropriatecontrol of the first and second stepper motors, the second slider 184(and the flipping cover assembly mounted thereon) can be translated inthe x- and y-directions relative to the frame (and hence the apparatushousing and the source and detector carried therein).

While FIGS. 22 and 23 do not show a z-motor, the skilled person willappreciate that the mechanism of FIGS. 22 and 23 may be provided with az-axis motor to enable the translation thereof in the z-direction.Alternatively, the source or appropriate optical components may beprovided with z-axis controls in a straightforward manner to permit therelative translation of a focal point relative to the sample in thez-direction, and realisation of the volumetric scanning abilitiesdiscussed herein.

In some cases, there will be an advantage in providing a mechanism tocalibrate the movement of the x-y-z motors to ensure traceability ofsample measurements. For example, in the pharmaceutical industry, “pillprofiling” is important to the pharmaceutical industry. Many pills areformulated to include a coating, which may be added for a variety ofreasons, such as taste masking, controlled or delayed release ordissolution, acid resistance in the stomach, and so on. For thesereasons accurate control of coating thickness is important. Determininghow the constituent ingredients of a pharmaceutical formulation aredistributed, arranged in layers, blended, and so on, is a crucial partof the formulation process and of the analysis of formulations.

The apparatuses of the invention enable samples to be analysed in athree-dimensional context, using the stepper motors to vary the point ofanalysis. However, for quality control purposes, it may be important tohave the measurement position calibrated.

Calibration can be enabled by providing a reference plinth manufacturedusing lithography e.g. from silicon with accurately manufacturedfeatures in the x-y plane for calibrating using the spectral maximum ofthe Raman signal from this substrate to calibrate the x-y motion of thestepper. Providing stepped features in this calibration plinth allowsone to calibrate the z motion. There are numerous materials that couldbe used to fabricate such a plinth. Interferometrically polished quartzwith evaporated Raman coatings are one example, though other methods ofmanufacture could be used to produce such a component to calibrate thestepper. The use of slip gauges could be developed to provide a quickcalibration of the z-drive movement increasing confidence inmeasurements mad eusing such apparatuses.

FIG. 24 is a sectional schematic view of a further apparatus, employingan alternative mechanism to bring the sample in position to the flippingcover of earlier embodiments.

The apparatus of FIG. 24 comprises a housing 200 shown in cut-away form,within which a spectrometer assembly 202 (which may be of any suitabletype and configuration, including replaceable or built-in) is mounted.The spectrometer assembly is capable of translation within the housingfrom a first (lower) position to a second (higher) position as indicatedby arrow 204. The assembly 202 houses a spectrometer having a source anda detector (not shown) and emits light 208 into an environmental chamberbody member 210 mounted on top of the spectrometer assembly 202.

A carrier 212 has a plinth removably mounted thereon to provide asample-receiving surface 214, which is similar to previous embodiments.The carrier is rotatable on a spindle 216 about an axis passing throughthe carrier. The spindle is mounted at one end on a centre post 218 inthe housing and on the other end by a roller 220 carried on a circulartrack 222 (albeit not appearing as truly circular in the drawing).Rotation of the carrier is achieved by a screw drive 224 and screwthread 226 at the top of the centre post 218. When the carrier isrotated the outer end travels around the circular track 222, and thecarrier body itself spins about the axis of the spindle.

A sample may be loaded on the carrier's sample-receiving surface 214,with the spectroscopic assembly 202 in the lowered position. The carriermay then be rotated until it is in position with the sample-receivingsurface facing downward and positioned over the environmental chamberbody member 210. The environmental chamber body member is then raised,and it makes a seal with the inverted carrier, so that thesample-receiving surface is enclosed and is exposed to the illuminationfrom the source within the thus-formed environmental chamber.

FIG. 25 is a perspective view of a sample holder (also called a dropheador plinth) 230, which has a circular drop-supporting surface dividedinto a major inner hydrophilic area 232 and a narrow annular hydrophobicband 234.

An aqueous droplet 236 is positioned on the sample-receiving surface andis confined to the hydrophilic area. The droplet has an outer coating238 of an oily composition which is immiscible with water and preventsevaporation, or may be used to study surface or interface phenomena andreactions. The boundary of the droplet is precisely pinned to thehydrophilic/hydrophobic boundary.

Alternatively, the droplet coating 238 and droplet body 236 may be apartially immiscible combination; this enable the study of thedissolution and diffusion of the cap liquid into the droplet, and canprovide valuable information about one or both liquids and theirinteraction.

There exist commercial pipetting technologies and perhaps the simplestof these developments pipettes two phases of liquid. Such dual pipettingis useful for the analysis of medical fluids where these can be pipettedonto the plinth in one operation. The second immiscible liquid wouldproduce a volume of sample trapped inside a cap of oil and this providesa sealed sample volume that does not evaporate and in which reactionkinetics can be studied in the bulk of the droplet, or at the oil-medialfluid interface. Such a sealed capped droplet sample could of course beproduced by first pipetting a sample and then using a smaller pipette todeliver the oil, lipid solution or other hydrophobic liquid phase. It ispossible of course to implement this embodiment as the converse withinner hydrophobic liquid on a hydrophobic area capped with a watersolution that rests on a hydrophilic outer ring. There are a growingnumber of microfluidic systems that can be adapted to deliver suchcomplex drop samples.

1. An apparatus for analyzing the optical properties of a sample,comprising: a housing to receive a light source and a detector inpredetermined positions; a sample locus defined relative to said housingand positioned such that when a light source and a detector are in saidpredetermined positions, the sample locus is subject to illumination bysaid light source and the detector is positioned to receive and detectlight from the sample; a cover on the housing, the cover being movablein a hinged manner between an open position and a closed position, thecover having an internal face which encloses the sample locus when thecover is in the closed position; a sample-receiving surface forreceiving a free-standing sample in liquid or semi-solid form, thesample-receiving surface being disposed on the internal face of thecover such that when the cover is in said open position thesample-receiving surface is exposed to and accessible by a user in agenerally horizontal orientation for loading of a sample thereon;wherein when the cover is moved to said closed position it encloses saidsample locus, with the sample-receiving surface being tilted away fromthe horizontal during said closing movement and the sample beingretained thereon by surface tension or adhesion and brought to saidsample locus in an enclosed environment.
 2. The apparatus of claim 1,wherein the sample-receiving surface is removable permitting itsreplacement by an alternative sample-receiving surface, or by aconventional sample holder into the position normally occupied by thesample-receiving surface.
 3. The apparatus of claim 2, wherein thesample holder is located in use within a hole in the cover, and when thesample holder is removed, the hole can be accessed from the externalcover surface to permit the insertion of an alternative sample holderfrom the rear side thereof.
 4. The apparatus of claim 1, wherein thesample-receiving surface is translated through at least 90 degrees asthe cover moves from the open position to the closed position.
 5. Theapparatus of claim 4, wherein the sample-receiving surface is translatedthrough approximately 180 degrees as the cover moves from the openposition to the closed position, with the sample-receiving surface beinginverted when the cover is closed.
 6. The apparatus of claim 1, whereinthe housing further comprises a support for receiving a spectroscopicassembly comprising a source and a detector mounted on a spectroscopicassembly housing, such that when said assembly is received by saidsupport, the source and detector are in said predetermined positions. 7.The apparatus of claim 1, wherein the source is coupled to an opticalfiber terminating at a terminating surface through which light iscoupled from the source to the sample locus, the terminating surfacebeing positioned such that when the cover is in a closed position, thesample is brought into contact with the terminating surface.
 8. Theapparatus of claim 1, wherein the detector is coupled to an opticalfiber terminating at a terminating surface through which light iscoupled from the sample locus to the detector, the terminating surfacebeing positioned such that when the cover is in a closed position, thesample is brought into contact with the terminating surface.
 9. Theapparatus of claim 7, wherein the terminating surface is provided on anend member carried on the end of said optical fiber, the end memberbeing transmissive to light of a frequency useful for spectroscopy. 10.The apparatus of claim 7, wherein both the source and the detector arecoupled to the same optical fiber having a single terminating surfacewhich couples light into and out of the sample.
 11. The apparatus ofclaim 7, wherein said terminating surface carries active materialthereon which enhances a spectral response.
 12. The apparatus of claim7, wherein the terminating surface is provided with a nanostructuredtreatment suitable to provide a surface enhanced Raman spectroscopy(SERS) response.
 13. The apparatus of claim 1, wherein the internalspace comprises a sample chamber which is enclosed when the cover is inthe closed position and which contains the sample locus, and aninstrumentation chamber which receives the source and detector, thesample chamber being separated from the instrumentation chamber by animpermeable wall having a wall section which is transparent to awavelength of radiation emitted by the source.
 14. The apparatus ofclaim 13, wherein the sample chamber is sealed when the cover is closed,and is sufficiently small in enclosed volume as to inhibit evaporationof a liquid sample droplet on said sample-receiving surface.
 15. Theapparatus of claim 13, wherein the sample chamber comprises a reservoirfor liquid whereby the enclosed volume becomes saturated with vapor fromsaid liquid when said cover is closed.
 16. The apparatus of claim 13,wherein the sample chamber is provided with one or more additionalenvironmental controls, such as a temperature control mechanism.
 17. Theapparatus of claim 1, further comprising a sensor adapted to detect whenthe cover reaches a predetermined position moving between the open andclosed positions, the sensor being operatively connected to a controlcircuit which prevents the source from being activated while the coveris open.
 18. The apparatus of claim 1, further comprising one or moremotors operable to displace the sample-receiving surface, when the coveris in the closed position, relative to the source and detector, therebypermitting fine adjustment of the material carried on thesample-receiving surface relative to the sample locus.
 19. The apparatusof claim 18, further comprising a motor controller operatively connectedto said one or more motors, the motor controller being operable to scanthe sample-receiving surface in a controlled manner relative to thesample locus.
 20. The apparatus of claim 18, wherein said one or moremotors include first and second motors which are disposed to displacethe sample-receiving surface in mutually orthogonal directions in theplane of the sample-receiving surface.
 21. The apparatus of claim 20,wherein said one or more motors include a third motor disposed todisplace the sample-receiving surface in a direction normal to the planeof the sample-receiving surface.
 22. The apparatus of claim 18, furthercomprising at least one additional motor operable to displace the sourceor an optical element through which the light travels from the source tothe sample locus, wherein the additional motor is operable to translatea focus of light from the source in a direction normal to the plane ofthe sample-receiving surface.
 23. The apparatus of claim 1, furthercomprising an optical sensing system to identify the boundaries of thesample.
 24. The apparatus of claim 23, further comprising a motorcontroller operatively connected to said one or more motors, the motorcontroller being operable to scan the sample-receiving surface in acontrolled manner relative to the sample locus, wherein said motorcontroller is programmed to perform a scan within the boundariesidentified by the optical sensing system. 25-28. (canceled)